scholarly journals Probing cathepsin K activity with a selective substrate spanning its active site

2003 ◽  
Vol 375 (2) ◽  
pp. 307-312 ◽  
Author(s):  
Fabien LECAILLE ◽  
Enrico WEIDAUER ◽  
Maria A. JULIANO ◽  
Dieter BRÖMME ◽  
Gilles LALMANACH
Keyword(s):  
Wild Type Mouse ◽  
Cathepsin K ◽  
Cysteine Proteases ◽  
Competitive Inhibitor ◽  
Mouse Fibroblast ◽  
Ionization State ◽  
Proline Residue ◽  
20 Nm ◽  
S2 Subsite ◽  
K Activity

The limited availability of highly selective cathepsin substrates seriously impairs studies designed to monitor individual cathepsin activities in biological samples. Among mammalian cysteine proteases, cathepsin K has a unique preference for a proline residue at P2, the primary determinant of its substrate specificity. Interestingly, congopain from Trypanosoma congolense also accommodates a proline residue in its S2 subsite. Analysis of a congopain model showed that amino acids forming its S2 subsite are identical with those of cathepsin K, except Leu67 which is replaced by a tyrosine residue in cathepsin K. Furthermore, amino acid residues of the congopain S2′ binding pocket, which accepts a proline residue, are strictly identical with those of cathepsin K. Abz-HPGGPQ-EDN2ph [where Abz represents o-aminobenzoic acid and EDN2ph (=EDDnp) represents N-(2,4-dinitrophenyl)-ethylenediamine], a substrate initially developed for trypanosomal enzymes, was efficiently cleaved at the Gly–Gly bond by cathepsin K (kcat/Km=426000 M−1·s−1). On the other hand, Abz-HPGGPQ-EDN2ph was resistant to hydrolysis by cathepsins B, F, H, L, S and V (20 nM enzyme concentration) and the Y67L (Tyr67→Leu)/L205A cathepsin K mutant (20 nM), but still acted as a competitive inhibitor. Taken together, the selectivity of Abz-HPGGPQ-EDN2ph to cathepsin K primarily depends on the S2 and S2′ subsite specificities of cathepsin K and the ionization state of histidine at P3. Whereas Abz-HPGGPQ-EDN2ph was hydrolysed by wild-type mouse fibroblast lysates, its hydrolysis was completely abolished in the cathepsin K-deficient samples, indicating that Abz-HPGGPQ-EDN2ph can be used to monitor selectively cathepsin K activity in physiological fluids and cell lysates.

scholarly journals S3 to S3' subsite specificity of recombinant human cathepsin K and development of selective internally quenched fluorescent substrates

2003 ◽  
Vol 373 (3) ◽  
pp. 981-986 ◽  
Author(s):  
Marcio F. M. ALVES ◽  
Luciano PUZER ◽  
Simone S. COTRIN ◽  
Maria Aparecida JULIANO ◽  
Luiz JULIANO ◽  
...  
Keyword(s):  
Amino Acids ◽  
Cathepsin K ◽  
Cysteine Proteases ◽  
Aminobenzoic Acid ◽  
Hydrophobic Amino Acids ◽  
Human Cathepsin ◽  
General Sequences ◽  
Natural Amino Acids ◽  
Positively Charged ◽  
K Activity

We have systematically examined the S3 to S3′ subsite substrate specificity requirements of cathepsin K using internally quenched fluorescent peptides derived from the lead sequence Abz-KLRFSKQ-EDDnp [where Abz is o-aminobenzoic acid and EDDnp is N-(2,4-dinitrophenyl)ethylenediamine]. We assayed six series of peptides, in which each position except Gln was substituted with various natural amino acids. The results indicated that the S3–S1 subsite requirements are more restricted than those of S1′–S3′. Cathepsin K preferentially accommodates hydrophobic amino acids with aliphatic side chains (Leu, Ile and Val) in the S2 site. Modifications at P1 residues also have a large influence on cathepsin K activity. Positively charged residues (Arg and Lys) represent the best accepted amino acids in this position, although a particular preference for Gly was found as well. Subsite S3 accepted preferentially basic amino acids such as Lys and Arg. A broad range of amino acids was accommodated in the remaining subsites. We further explored the acceptance of a Pro residue in the P2 position by cathepsin K in order to develop specific substrates for the enzyme. Two series of peptides with the general sequences Abz-KXPGSKQ-EDDnp and Abz-KPXGSKQ-EDDnp (where X denotes the position of the amino acid that is altered) were synthesized. The substrates Abz-KPRGSKQ-EDDnp and Abz-KKPGSKQ-EDDnp were cleaved by cathepsin K at the Arg–Gly and Gly–Ser bonds respectively, and have been shown to be specific for cathepsin K when compared with other lysosomal cysteine proteases such as cathepsins L and B and with the aspartyl protease cathepsin D.

c-Jun Inhibits Thapsigargin-Induced ER Stress Through Up-Regulation of DSCR1/Adapt78

2008 ◽  
Vol 233 (10) ◽  
pp. 1289-1300 ◽  
Author(s):  
Peng Zhao ◽  
Xiaoyan Xiao ◽  
Agnes S. Kim ◽  
M. Fatima Leite ◽  
Jinxia Xu ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Cell Death ◽  
Er Stress ◽  
Wild Type Mouse ◽  
Mouse Fibroblast ◽  
Expression Levels ◽  
Mouse Fibroblasts ◽  
The Unfolded Protein Response ◽  
Calcineurin Activity

The endoplasmic reticulum (ER) is exquisitely sensitive to changes in its internal environment. Various conditions, collectively termed “ER stress”, can perturb ER function, leading to the activation of a complex response known as the unfolded protein response (UPR). Although c-Jun N-terminal kinase (JNK) activation is nearly always associated with cell death by various stimuli, the functional role of JNK in ER stress-induced cell death remains unclear. JNK regulates gene expression through the phosphorylation and activation of transcription factors, such as c-Jun. Here, we investigated the role of c-Jun in the regulation of ER stress-related genes. c-Jun expression levels determined the response of mouse fibroblasts to ER stress induced by thapsigargin (TG, an inhibitor of sarco/endoplasmic reticulum Ca2+ ATPase). c-jun−/− mouse fibroblast cells were more sensitive to TG-induced cell death compared to wild-type mouse fibroblasts, while reconstitution of c-Jun expression in c-jun−/− cells (c-Jun Re) enhanced resistance to TG-induced cell death. The expression levels of ER chaperones Grp78 and Gadd153 induced by TG were lower in c-Jun Re than in c-jun−/− cells. Moreover, TG treatment significantly increased calcineurin activity in c-jun−/− cells, but not in c-Jun Re cells. In c-Jun Re cells, TG induced the expression of Adapt78, also known as the Down syndrome critical region 1 (DSCR1), which is known to block calcineurin activity. Taken together, our findings suggest that c-Jun, a transcription factor downstream of the JNK signaling pathway, up-regulates Adapt78 expression in response to TG-induced ER stress and contributes to protection against TG-induced cell death.

scholarly journals Blockade of the sympathetic nervous system degrades ligament in a rat MCL model

2004 ◽  
Vol 96 (2) ◽  
pp. 711-718 ◽  
Author(s):  
Kelley W. Dwyer ◽  
Paolo P. Provenzano ◽  
Peter Muir ◽  
Wilmot B. Valhmu ◽  
Ray Vanderby
Keyword(s):  
Nervous System ◽  
Sympathetic Nervous System ◽  
Plasma Concentrations ◽  
Cathepsin K ◽  
Cysteine Proteases ◽  
Collagenolytic Activity ◽  
Tartrate Resistant Acid Phosphatase ◽  
Real Time Quantitative Pcr ◽  
Osmotic Pumps ◽  
Sympathetic Nervous

We hypothesize that blockade of the sympathetic nervous system degrades ligament. We tested this hypothesis in a rat medial collateral ligament (MCL) model. Fifteen animals were treated for 10 days with the sympathetic chemotoxin guanethidine using osmotic pumps, whereas 15 control rats received pumps containing saline. A reduction in plasma concentrations of norepinephrine in the guanethidine rats indicated a significant decrease in sympathetic nerve activity. Vasoactive intestinal peptide and neuropeptide Y were decreased in MCLs from guanethidine animals, as quantified by radioimmunoassays. Tissue vascularity was substantially increased in guanethidine MCLs, whereas mechanical properties were significantly decreased. Proteases, such as matrix metalloproteinases (MMP) and cysteine proteases, play a major role in ligament degradation. The proteases MMP-13, cathepsin K, and tartrate-resistant acid phosphatase (TRAP) have collagenolytic activity and have been shown in rat ligament tissues. To determine whether the degradation seen in this study was due to protease activity, we determined the expression of these enzymes in control and treated MCLs. Real-time quantitative PCR revealed that guanethidine treatment increased expression of MMP-13 and cathepsin K mRNAs, although overall expression levels of MMP-13 and TRAP were relatively low. Histology also identified increases in TRAP and cathepsin K, but not MMP-13, in guanethidine-treated tissues. Results support our hypothesis that blockade of the sympathetic nervous system substantially degrades ligament.

Fluorescence Microscopic Demonstration of Cathepsin K Activity as the Major Lysosomal Cysteine Proteinase in Osteoclasts

1998 ◽  
Vol 123 (4) ◽  
pp. 752-759 ◽  
Author(s):  
T. Kamiya ◽  
Y. Kobayashi ◽  
K. Kanaoka ◽  
T. Nakashima ◽  
Y. Kato ◽  
...  
Keyword(s):  
Cysteine Proteinase ◽  
Cathepsin K ◽  
Microscopic Demonstration ◽  
K Activity

Diagnostic possibilities of laboratory assessment of cathepsin K activity in gingival and peri-implant fluid under normal conditions and periimplantitis

Stomatologiya ◽  
2021 ◽  
Vol 100 (3) ◽  
pp. 55
Author(s):  
O.A. Zorina ◽  
M.A. Amkhadova ◽  
O.A. Boriskina ◽  
E.Sh. Aleskerov ◽  
V.M. Polyakov
Keyword(s):  
Cathepsin K ◽  
Laboratory Assessment ◽  
K Activity

Double Knockouts Reveal That Protein Tyrosine Phosphatase 1B Is a Physiological Substrate of Calpain-1 in Platelets.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 396-396 ◽  
Author(s):  
Shafi M. Kuchay ◽  
William P. Fay ◽  
Athar H. Chishti
Keyword(s):  
Platelet Aggregation ◽  
Protein Tyrosine Phosphatase ◽  
Tyrosine Phosphatase ◽  
Wild Type Mouse ◽  
Cysteine Proteases ◽  
Protein Tyrosine Phosphatase 1B ◽  
Dependent Manner ◽  
Wild Type ◽  
Clot Retraction ◽  
Protein Tyrosine

Abstract Calpains are ubiquitous calcium-regulated cysteine proteases that have been implicated in cytoskeletal organization, cell proliferation, apoptosis, cell motility, and hemostasis. Previously we used gene-targeting to evaluate the physiological function of mouse calpain-1, and established that its inactivation results in reduced platelet aggregation and clot retraction, potentially by causing dephosphorylation of platelet proteins. Here, we present data showing that calpain-1 null platelets accumulate protein tyrosine phosphatase 1B (PTP1B) that correlates with enhanced tyrosine phosphatase activity and dephosphorylation of multiple substrates in platelets. Using antibodies specific for phosphotyrosines 747 and 759 of the b3 subunit of αIibβ3 integrin, we show that the tyrosine phosphorylation of both tyrosine residues at positions 747 and 759 in the cytoplasmic domain of b3 subunit is reduced by approximately 60–70% in the calpain-1 null platelets. Treatment of calpain-1 null platelets with DMHV, an inhibitor of tyrosine phosphatases, corrected the aggregation defect and recovered impaired clot retraction. Importantly, platelet aggregation, clot retraction, and tyrosine dephosphorylation defects were rescued in the double knockout mice lacking both calpain-1 and PTP1B. Consistent with this paradigm, treatment of wild type mouse platelets as well as human platelets with the tyrosine phosphatase inhibitor DMHV enhanced their aggregation at low doses of thrombin. Conversely, MDL, a cell permeable inhibitor of calpains, potently inhibited aggregation of wild type mouse platelets in a dose-dependent manner upon thrombin activation. Further evaluation of mutant mice by ferric chloride induced arterial injury model suggests that the calpain-1 null mice are relatively resistant to thrombosis in vivo. Finally, the calpain-1 mediated regulation of PTP1B appears to be a systemic event as evident by the enhanced tyrosine dephosphorylation of B lymphocytes and their resistance to apoptosis in calpain-1 null mice. Together, our results demonstrate that PTP1B is a physiological substrate of calpain-1 and suggest that a similar mechanism may regulate calpain-1 mediated tyrosine dephosphorylation in other cells.

scholarly journals Expression and alteration of the S2 subsite of the Leishmania major cathepsin B-like cysteine protease

1999 ◽  
Vol 340 (1) ◽  
pp. 113-117 ◽  
Author(s):  
Victor J. CHAN ◽  
Paul M. SELZER ◽  
James H. McKERROW ◽  
Judy A. SAKANARI
Keyword(s):  
Cathepsin B ◽  
Leishmania Major ◽  
Cathepsin L ◽  
Expression System ◽  
Specific Inhibitor ◽  
Structural Similarity ◽  
Cysteine Proteases ◽  
Recombinant Enzyme ◽  
Yeast Expression System ◽  
S2 Subsite

The mature form of the cathepsin B-like protease of Leishmania major (LmajcatB) is a 243 amino acid protein belonging to the papain family of cysteine proteases and is 54% identical to human-liver cathepsin B. Despite the high identity and structural similarity with cathepsin B, LmajcatB does not readily hydrolyse benzyloxycarbonyl-Arg-Arg-7-amino-4-methyl coumarin (Z-Arg-Arg-AMC), which is cleaved by cathepsin B enzymes. It does, however, hydrolyse Z-Phe-Arg-AMC, a substrate typically cleaved by cathepsin L and B enzymes. Based upon computer generated protein models of LmajcatB and mammalian cathepsin B, it was predicted that this variation in substrate specificity was attributed to Gly234 at the S2 subsite of LmajcatB, which forms a larger, more hydrophobic pocket compared with mammalian cathepsin B. To test this hypothesis, recombinant LmajcatB was expressed in the Pichia pastoris yeast expression system. The quality of the recombinant enzyme was confirmed by kinetic characterization, N-terminal sequencing, and Western blot analysis. Alteration of Gly234 to Glu, which is found at the corresponding site in mammalian cathepsin B, increased recombinant LmajcatB (rLmajcatB) activity toward Z-Arg-Arg-AMC 8-fold over the wild-type recombinant enzyme (kcat/Km = 3740±413 M-1·s-1 versus 472±72.4 M-1·s-1). The results of inhibition assays of rLmajcatB with an inhibitor of cathepsin L enzymes, K11002 (morpholine urea-Phe-homoPhe-vinylsulphonylphenyl, kinact/Ki = 208200±36000 M-1·s-1), and a cathepsin B specific inhibitor, CA074 [N-(L-3-trans-propylcarbamoyloxirane-2-carbonyl)-ʟ-isoleucyl-L-proline, kinact/Ki = 199200±32900 M-1·s-1], support the findings that this protozoan protease has the P2 specificity of cathepsin L-like enzymes while retaining structural homology to mammalian cathepsin B.

Analysis of Cathepsin-K Activity at Tooth and Dental Implant Sites and the Potential of This Enzyme in Reflecting Alveolar Bone Loss

2012 ◽  
Vol 83 (4) ◽  
pp. 498-505 ◽  
Author(s):  
Nermin Yamalik ◽  
Sevim Günday ◽  
Serdar Uysal ◽  
Kamer Kilinç ◽  
Erdem Karabulut ◽  
...  
Keyword(s):  
Bone Loss ◽  
Dental Implant ◽  
Alveolar Bone ◽  
Cathepsin K ◽  
Alveolar Bone Loss ◽  
K Activity

scholarly journals Kinetic analysis of the effects of glycosaminoglycans and lipoproteins on urokinase-mediated plasminogen activation

1991 ◽  
Vol 276 (3) ◽  
pp. 785-791 ◽  
Author(s):  
J M Edelberg ◽  
M Weissler ◽  
S V Pizzo
Keyword(s):  
Kinetic Analysis ◽  
Catalytic Domain ◽  
Dissociation Constants ◽  
Low Density Lipoprotein ◽  
Heparan Sulphate ◽  
Density Lipoprotein ◽  
Competitive Inhibitor ◽  
Plasminogen Activation ◽  
20 Nm

The glycosaminoglycans (GAGs) heparin, heparan sulphate and chondroitin 6-sulphate stimulate the rate of urokinase activation of human plasminogen. Kinetic analysis of plasminogen activation demonstrates that heparin, heparan sulphate and chondroitin 6-sulphate increased the catalytic rate (Kcat) by 5.3-, 3.5- and 2.5-fold respectively. These stimulatory GAGs had no effect on the affinity of urokinase for plasminogen, since the Km of the reaction is unaltered by the GAGs. The GAGs may enhance the rate of plasminogen activation through an interaction with the catalytic domain of the urokinase, with dissociation constants of approx. 30 nM. Additionally, the lipoproteins, lipoprotein (a) [Lp(a)] and low-density lipoprotein (LDL) inhibit heparin and heparan sulphate stimulation of plasmin formation. Lp(a) is a competitive inhibitor (Kic 20 nM) and LDL is a mixed inhibitor of heparin-enhanced urokinase-mediated plasminogen activation (Kic 24 nM and Kiu 60 nM). These inhibition constants correlate with physiological concentrations of these lipoproteins. These data suggest that these GAGs and lipoproteins may play an important role in vivo in regulating urokinase-mediated plasmin formation.

scholarly journals Cathepsin K and matrix metalloproteases activity in bone tissue of OXYS rats with osteoporosis development

2010 ◽  
Vol 56 (2) ◽  
pp. 274-282 ◽  
Author(s):  
A.A. Venediktova ◽  
O.V. Falameeva ◽  
N.G. Kolosova ◽  
M.A. Sadovoj ◽  
T.A. Korolenko
Keyword(s):  
Bone Tissue ◽  
Wistar Rats ◽  
Cathepsin K ◽  
Typical Feature ◽  
Matrix Metalloproteases ◽  
Oxys Rats ◽  
Age Related ◽  
Α2 Macroglobulin ◽  
The Comparative Study ◽  
K Activity

The comparative study of аctivity of cysteine protease cathepsin K and matrix metalloproteases (MMPs) in bone tissue of accelerated senescent OXYS rats with early ageing comparatively to Wistar rats of the same age was performed. Early development osteoporosis is a typical feature of OXYS rats. In bone tissue of 3 month old OXYS rats, before appearance of osteoporosis manifestation cathepsin K activity was higher, whereas MMPs activity was lower than in Wistar rats. In Wistar rats (3 and 14 months old) cathepsin K activity of spine was shown to increase, and MMPs activity to decrease. In OXYS rats age-related change of cathepsin K and MMPs activity in bone tissue had the opposite direction. As a result of this there were no differences between Wistar and OXYS rats 14 months old despite the marked osteoporosis in OXYS rats as revealed our early researches. Serum α2-macroglobulin activity was higher in 14 months old OXYS rats. The role of activation of cathepsin K in bone resorption in the development of osteoporosis in early ageing OXYS rats is discussed.

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